Vegetative salt tolerance of barnyard grass mutants selected for salt tolerant germination

Improving salt tolerance of economically important plants is imperative to cope with the increasing soil salinity in many parts of the world. Mutation breeding has been widely used to improve plant performance under salinity stress. In this study, we have mutagenized Echinochloa crusgalli L. with sodium azide and three selected mutants (designated fows A) with salt tolerant germination. Their vegetative growth was compared to that of the wild type after short-term and long-term salt stress. The germination of the three fows A mutants in the presence of inhibitory concentrations of NaCl, KCL, and mannitol was better than that of the wild type. Early growth of the mutants in the presence of 200 mM NaCl was also better than that of the wild type perhaps due to improved K⁺ uptake and enhanced accumulation of sugars particularly sucrose at least in two mutants. But the three mutants and the wild type responded similarly to long-term salt stress. The tolerance mechanisms during short-term and long-term salt stress are discussed.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance

Detrimental effects of salinity on plants are known to be partially alleviated by external Ca²⁺. Previous work demonstrated that the Arabidopsis SOS3 locus encodes a Ca²⁺‐binding protein with similarities to CnB, the regulatory subunit of protein phosphatase 2B (calcineurin). In this study, we further characterized the role of SOS3 in salt tolerance. We found that reduced root elongation of sos3 mutants in the presence of high concentrations of either NaCl or LiCl is specifically rescued by Ca²⁺ and not Mg²⁺, whereas root growth is rescued by both Ca²⁺ and Mg²⁺ in the presence of high concentrations of KCl. Phenocopies of sos3 mutants were obtained in wild‐type plants by the application of calmodulin and calcineurin inhibitors. These data provide further evidence that SOS3 is a calcineurin‐like protein and that calmodulin plays an important role in the signalling pathways involved in plant salt tolerance. The origin of the elevated Na : K ratio in sos3 mutants was investigated by comparing Na⁺ efflux and influx in both mutant and wild type. No difference in Na⁺ influx was recorded between wild type and sos3; however, sos3 plants showed a markedly lower Na⁺ efflux, a property that would contribute to the salt‐oversensitive phenotype of sos3 plants.     

向日葵E3泛素连接酶基因的分析定位和表达鉴定

该研究利用前期获得的向日葵耐盐相关基因E3泛素连接酶基因序列(HERC2),构建瞬时表达载体Cam-35S-HERC2-GFP,采用基因枪法转化洋葱表皮细胞进行亚细胞定位;采用RT-PCR技术,分析盐胁迫下HERC2在耐盐品种P50和盐敏感品种P29根、下胚轴和叶中的表达差异;构建HERC2植物表达载体pPZP221-HERC2,采用农杆菌介导法将HERC2导入烟草,进行耐盐功能验证。结果表明:(1)HERC2蛋白定位在细胞膜、细胞质和细胞核中。(2)受到NaCl胁迫后,HERC2基因在耐盐品种P50和盐敏感品种P29中均上调表达,但耐盐品种中的表达量较高。(3)HERC2基因的表达,能够提高转基因烟草的耐盐性。该研究结果为进一步解析向日葵对盐胁迫的响应机制,以及耐盐新品种的选育奠定了基础。     

Differential salt-stress response during germination and vegetative growth in in vitro selected somaclonal mutants of Cenchrus ciliaris L.

Four somaclonal mutants (S1, S4, S6 and M10) and their parental Cenchrus ciliaris L. cultivar Biloela were characterized under salinity conditions at germination and vegetative growth stages. Seeds of all somaclonal mutants had higher germination percentages than cv. Biloela seeds in the control and salt treatments. At 150 mM, germination was significantly higher in M10, S6 and S4 (72.3%, 66.3% and 61.8%, respectively) than in cv. Biloela (35.5%). Mutants grown under salinity along with cv. Biloela for 35 days had a different relative growth rate. S6 had the highest growth rate, indicating its potential tolerance to salt stress, whereas M10 was the most sensitive, with Bi, S4 and S1 being intermediate tolerant genotypes. Catalase enzyme activity (CAT) in M10 decreased in response to salt stress and was significantly associated with malondialdehide content, suggesting salt injury, whereas higher levels of CAT activity in S6 during salt stress were associated with increased salinity tolerance. The present results indicate that somaclonal variation and in vitro mutagenesis offer an effective tool for improvement of C. ciliaris because the somaclonal mutants showed differential tolerance to salt stress with respect to their parental and could be a better choice for use in a breeding program.     

Comparative study on seed germination characteristics of two species of Australia saltbush under salt stress

In order to examine the response characteristics and possible reasons of Atriplex lentiformis and Atriplex undulata under salt stress at stage of seed germination, the seeds were treated with different concentrations of NaCl (0, 50, 100, 200 and 300 mmol⋅L⁻¹), 20 mmol⋅L⁻¹ LiCl or mannitol whose iso-osmotic concentrations corresponding to 200 mmol⋅L⁻¹ NaCl. The results showed that the germination rate of two species of saltbush was depressed with the increase of NaCl concentration, and A. lentiformis showed greater salt tolerance compared with A. undulata. After removal of salt stress, the final germination ratio of A. lentiformis was over 93%, while that of A. undulata was only 56%. Evans blue staining revealed that 200 mmol⋅L⁻¹ NaCl did not damage membrane permeability of A. lentiformis seed embryos, but significantly increased the membrane permeability of A. undulata seed embryos and caused irreversible damage to them, especially radicles. The results on water uptake indicated that the inhibition of NaCl on seed germination was mainly due to osmotic stress instead of ionic toxicity, and A. lentiformis exhibited higher salt tolerance due to its greater resistance to osmotic stress.     

Mutation induced by ethylmethanesulphonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (<Emphasis Type="Italic">Ipomoea</Emphasis><Emphasis Type="Italic">batatas</Emphasis> L.)

Salt tolerant cultivars of sweet potato (Ipomoea batatas L.) can be obtained from induced mutation. The objective of the present study was to induce mutation for salt tolerance using ethylmethanesulphonate (EMS) in calli of sweet potato, followed by cell line selection and subsequent plant regeneration. Calli initiated from leaf explants were treated with 0.5% EMS for 0, 1, 1.5, 2, 2.5 and 3 h, followed by rinsing with sterile distilled water for four times. Preliminary experiments showed that 200 mM NaCl could be used as selection pressure. Salt tolerant calli were sub-cultured on medium supplemented with 200 mM NaCl for selection of mutant cell lines and this process repeated 5 times (20 days each). The selected calli were transferred onto somatic embryo formation medium, which was Murashige and Skoog (MS) medium supplemented with 4 mg l⁻¹ abscisic acid (ABA), 10 mg l⁻¹ gibberellic acid (GA). After 15 days, somatic embryos were transferred onto MS medium supplemented with 0.05 mg l⁻¹ ABA, 0.2 mg l⁻¹ zeatin (ZT) for regeneration. Plants designated as ML1, ML2 and ML3 were regenerated from the somatic embryos formed by calli treated with 0.5% EMS for 2 and 2.5 h. After propagation, salt tolerance of these mutants was investigated. Data suggested the mutants were more salt tolerant than control plants.     

Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K(+)/Na(+) selectivity and proline accumulation

The eco-physiology of salt tolerance, with an emphasis on K⁺ nutrition and proline accumulation, was investigated in the halophyte Thellungiella halophila and in both wild type and eskimo-1 mutant of the glycophyte Arabidopsis thaliana, which differ in their proline accumulation capacity. Plants cultivated in inert sand were challenged for 3 weeks with up to 500 mM NaCl. Low salinity significantly decreased A. thaliana growth, whereas growth restriction was significant only at salt concentrations equal to or exceeding 300 mM NaCl in T. halophila. Na⁺ content generally increased with the amount of salt added in the culture medium in both species, but T. halophila showed an ability to control Na⁺ accumulation in shoots. The analysis of the relationship between water and Na⁺ contents suggested an apoplastic sodium accumulation in both species; this trait was more pronounced in A. thaliana than in T. halophila. The better NaCl tolerance in the latter was associated with a better K⁺ supply, resulting in higher K⁺/Na⁺ ratios. It was also noteworthy that, despite highly accumulating proline, the A. thaliana eskimo-1 mutant was the most salt-sensitive species. Taken together, our findings indicate that salt tolerance may be partly linked to the plants’ ability to control Na⁺ influx and to ensure appropriate K⁺ nutrition, but is not linked to proline accumulation.     

Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance

• There is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. We hypothesized that bacteria from the phyllosphere of these plants might play a key role in its high tolerance to excessive salinity.
• Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant‐growth promoting (PGP) properties, enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied.
• Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na⁺ in its shoots, thus improving sodium phytoextraction capacity.
• Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations. The combined use of A. macrostachyum and its microbiome can be an adequate tool to enhance plant adaptation and sodium phytoextraction during restoration of salt degraded soils.

     

The aba3-1 Mutant of Arabidopsis thaliana Withstands Moderate Doses of Salt Stress by Modulating Leaf Growth and Salicylic Acid Levels

The role of abscisic acid (ABA) and salicylic acid (SA) in salt stress tolerance was studied in Arabidopsis thaliana using mutants that show a defect in hormone biosynthesis or signaling. Plants were subjected to either control conditions (irrigated with nutrient solution) or a moderate salt stress (nutrient solution + 100 mM NaCl), and the response of the aba3, abi4, sid2, and eds5 mutants (with defective ABA or SA biosynthesis/signaling) was compared to that of the wild type (WT). A particular phenotype was observed in the aba3 mutant, which was characterized by reduced plant biomass and lower relative leaf water contents (RWC) under control conditions. However, salt stress reduced growth in the WT, sid2, and eds5 mutants, and to a lesser extent in the abi4 mutant, but not in the aba3 mutant. An analysis of the hormonal balance of leaves revealed that altered SA levels may explain, at least partly, growth changes in the aba3 mutant, under both control and salt stress conditions. The aba3-1 mutant showed higher SA levels than the WT under control conditions and a drastic decrease in the levels of this plant growth regulator under salt stress, an aspect that was not observed in the WT. However, reductions in endogenous SA levels in sid2 and eds5 mutants did not result in increased growth either under control or salt stress conditions. Among the tested genotypes, the aba3 mutant was the only one in which jasmonic acid (JA) levels did not increase in response to salt stress. It is concluded that although ABA deficiency can severely affect plant growth and water relations in aba3 mutants, these plants modulate, among other processes, leaf growth and SA levels, which help them withstand moderate doses of salt stress.     

转CkLEA4基因拟南芥种子萌发期的抗逆性分析

该研究在实验室前期研究的基础上,将受脱水、盐胁迫和ABA诱导的柠条锦鸡儿CkLEA4基因转入野生型拟南芥,并利用实时荧光定量PCR从8株纯合体中筛选出3个表达量不同的株系,比较野生型和转CkLEA4基因过表达拟南芥种子在不同胁迫处理下的萌发率,以探讨CkLEA4基因在植物抵抗逆境胁迫中的功能。结果发现:(1)在不同浓度NaCl、甘露醇及ABA处理下,转CkLEA4基因过表达拟南芥种子的萌发率均高于野生型,随着NaCl、甘露醇及ABA浓度增加,各株系萌发率均降低,但野生型的萌发率下降幅度均高于3个过表达株系,并且在200mmol/L NaCl和400mmol/L甘露醇处理下,过表达株系子叶绿化率均显著高于野生型。(2)在低浓度ABA处理下,CkLEA4过表达植株子叶的绿化率也高于野生型。研究表明,柠条锦鸡儿CkLEA4基因提高了拟南芥种子萌发阶段对盐、ABA及渗透胁迫的耐受性。     

Extracts of the Brown Alga Dictyota dichotoma (Hudson) J.V. Lamouroux Alleviate Salt Stress in Rice (Oryza sativa L.) During Germination

Algal extracts provide a safe regime for enhancing crop productivity under stressful conditions. The present study evaluated the efficiency of aqueous and ethanolic extracts of the brown alga Dictyota dichotoma in alleviation of salt stress on germination of rice seeds. Firstly, seeds were germinated using the aqueous extract of D. dichotoma at concentrations of 0, 5, 10, 20, and 50 g L⁻¹, prepared either at room temperature (RTE) or by boiling (BLE). The % germination of rice increased from 84% in non-treated seeds to 100% when treated with 20 g L⁻¹ BLE, although this treatment caused reduced uniformity of germination. Embryo growth was maximum at 20 g L⁻¹ of both extracts with superiority of BLE over RTE. In the second experiment, the effect of 20 g L⁻¹ aqueous and ethanolic extracts relative to a balanced nutrient supply on germination of seeds treated with 0, 40, 90, and 170 mM NaCl was investigated. Salinity reduced % germination with delayed onset but high uniformity of germination, whereas algal amendments counterbalanced the effect of salinity to a greater extent relative to the nutrient supply. Upon withdrawal of salt stress, seeds promptly recovered, with more efficient recovery of seeds exposed to 170 mM than from 90 mM NaCl. The lower recovery of salt-treated seeds compared with the control seed germination suggests that rice suffered from the toxic ion effect of salinity on embryo rather than from the osmotic effect. Extracts of D. dichotoma can enhance and also alleviate salinity stress on rice seed germination.

     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.     

In vivo evolutionary engineering of a boron-resistant bacterium: <Emphasis Type="Italic">Bacillus boroniphilus</Emphasis>

Boron is an industrially and biologically important element. However, the mechanisms of boron tolerance and its transport in bacteria and many other living systems are still not clearly understood. In this study, the boron resistance level of a boron-tolerant bacterium, Bacillus boroniphilus DSM 17376, was improved up to 300 mmol l⁻¹ boron, by employing an in vivo evolutionary engineering strategy based on batch selection under continuous exposure to gradually increasing boron stress levels. The resistance was heterogeneous within the final mutant population which ranged from about 1- to 16-fold of the wild type resistance at 150 mmol l⁻¹ boron stress level. Boron-resistant mutants had significant cross-resistance to iron and copper stresses, and were also cross-resistant to salt (NaCl) stress, suggesting a common resistance mechanism between these stress types. Additionally, highly boron-resistant mutants had up to 2.8-fold higher boron contents than the wild-type, when exposed to high levels of (150 mmol l⁻¹) continuous boron stress throughout their cultivation. It was shown that evolutionary engineering is a successful approach to significantly increase bacterial boron resistance and investigate the complex mechanism of boron tolerance and transport in microbial systems.     

水稻资源芽期和苗期耐盐碱性综合评价及耐盐基因分析

培育耐盐碱水稻品种是应对全球人口日益增长的重要途径之一。文中以21份耐盐碱性不同的水稻品种(系)为材料,在芽期和苗期设置6个不同盐碱浓度处理,测定了发芽势、发芽率、芽长、根长、根数、芽鲜重和苗鲜总重等指标,以各指标盐害率的平均值作为耐盐碱性的综合评价标准。结果表明随着盐碱浓度的提升,对种子萌发和生长的抑制越明显。在1%NaCl加0.25%NaHCO3溶液处理下,发芽率盐害率变异最大,为0%–89.80%。在所有浓度处理下,各性状指标的盐害率都具有相似的变化趋势。筛选到4份综合耐盐碱能力强(大酒谷、日本晴、魔王谷和02428)和7份弱的种质资源。比较了4份耐盐碱强和3份耐盐碱弱的资源耐盐基因序列:OsHAL3和OsRR22基因在7份材料中没有差异,SKC1和DST基因在耐盐碱强和耐盐碱弱的品种之间有明显的变异。研究结果为进一步挖掘水稻耐盐碱基因和培育耐盐碱水稻新品种提供了种质资源和理论基础。     

A novel wheat α‐amylase inhibitor gene,TaHPS, significantly improves the salt and drought tolerance of transgenic Arabidopsis

On the basis of microarray analyses of the salt‐tolerant wheat mutant RH8706‐49, a previously unreported salt‐induced gene, designated as TaHPS [Triticum aestivum hypothetical (HPS)‐like protein], was cloned. Real‐time quantitative polymerase chain reaction analyses showed that expression of the gene was induced by abscisic acid, salt and drought. The encoded protein was found to be localized mainly in the plasma membranes. Transgenic Arabidopsis plants overexpressing TaHPS were more tolerant to salt and drought stresses than non‐transgenic wild‐type (WT) plants. Under salt stress, the root cells of the transgenic plants secreted more Na⁺ and guard cells took up more Ca²⁺ ions. Compared with wild‐type plants, TaHPS‐expressing transgenic plants showed significantly lower amylase activity and glucose and malic acid levels. Our results showed that the expression of TaHPS inhibited amylase activity, which subsequently led to a closure of stomatal apertures and thus improved plant tolerance to salt and drought.     

Arabidopsis mutants with reduced response to NaCl and osmotic stress

We isolated 6 mutant lines of Arabidopsis thaliana that expressed reduced sensitivity to salt and osmotic stress during germination. All 6 lines cum recessive mutations in a single gene, designated reduced salt sensitivity (rss), linked to the ADH marker on chromosome 1. The rss mutants are less sensitive than wild type for NaCl and osmotic stress inhibition of germination, tolerating approximately 150 mM higher concentrations of NaCl and about 250 mM higher concentrations of sorbitol in the media. Germination assays on media containing various salts indicate that the rss mutations reduce sensitivity lo Na⁺ and Rh⁺ but also, to a much lesser degree, to K⁺ and Cs^s+. However, the rss mutation does not improve plant growth when plantlets are transferred to high salt or high osmotic pressure media after germination. The rss plantlets accumulate praline to a significantly lesser degree than wild type when they are exposed to either salt or osmotic stress. Thus, the rss mutants differ from wild type both at germination and during vegetative growth indicating that the rss mutations are pleiotropic and might affect perception of solutes or some aspect of stress-induced signaling. The rss mutations do not alter ABA sensitivity and therefore probably do not affect ABA-mediated signaling.